| Research Abstract |
To clarify the effect of thrombin on the production of reactive oxygen species (ROS) in vasular endothelial cells, we analyzed intracellular amount of hydrogen peroxide in cultured human umbilical vein endothelial cells (HUVEC) using the fluorescence probe (DCFH-DA). In HUVEC, ROS was increased up to two-folds of control by the treatment of 1U/ml thrombin for 24 hours. Carbonyl cyanide m-chlorophenylhyazone, an agent for decreasing the mitochondrial membrane proton gradient and thenoyltrifluoroacetone, a complex II inhibitor, markedly suppressed the overproduction of ROS in thrombin-treated HUVEC. Quinacrine, an inhibitor for NADPH oxidase, also significantly suppressed the overproduction of ROS. In contrast, no significant inhibitory effects of indomethacin, an inhibitor for cyclooxygenase, or of L-NAME, an inhibitor for NO synthase, were observed. DPI, an inhibitor for complex I, and myxothiazol, an inhibitor for complex III, also did not exhibit a significant effect on the production of ROS. Con focal laser microscopy imaging showed that green fluoresscence of DCFH-DA colocalized with red fluorescence of Mitotracker Red, indicating hydrogen peroxide production from mitochondria. Pre-treatment of aspirin (1mM) markedly suppressed the ROS production in thrombin-treated HUVEC.
These findings showed that thrombin could cause the overproduction of ROS from mitochondrial respiratory chain and NADPH oxidase, and subsequently the consumption of NO in vascular endothelial cells, leading to injury of vscular endothelial cells. In addition, aspirin may protect vascular endothelial cells from damage by trombin-induced ROS poduction.
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